It has been established that Mycobacterium bovis BCG cell walls and Mycobacterium phlei cell walls attached to minute oil droplets possess potent antitumor activity in an experimental animal model, and these preparations are in the initial stages of clinical investigation. Components of these cell walls that are effective in tumor regression, however, have not been identified and characterized. The overall objective of this proposed research is to isolate and chemically characterize these components, in order to aid in the development of mycobacterial components as immunotherapeutic agents for cancer. Components of the cell walls that are required for antitumor activity will be identified by testing the cell wall at each stage of a systematic fractionation protocol designed to extract components of predictable chemical properties. The various extracts will be further fractionated, and individual components will be combined one at a time with the remaining insoluble "cell wall skeletons" and examined for antitumor activity. It is now known that organic solvent extraction of the cell wall greatly reduces its antitumor activity, and one component in the extract, a glycolipid designated P3, has been found to restore the tumor-regressive activity of the extracted cell wall. The structure of P3 has not been completely determined. The other components in these extracts have not been carefully purified and retested for activity. One such component, a 3-0-methylglucose-containing glycolipid (MGG), has been found in mycobacterium species for the first time. P3, MGG, a newly isolated acidic arabinomannan lipopolysaccharide, and other extractable mycobacterial cell wall components will be characterized chemically, and their biological and immunological properties will be determined. Purification of these components will utilize the techniques of high pressure liquid chromatography, gel column chromatography, and ion exchange column chromatography, and their characterization will utilize high resolution mass spectrometry, combined gas chromatography mass spectrometry, chemical ionization mass spectrometry, nuclear magnetic resonance spectrometry, immunochemistry, and classical as well as newly developed techniques of lipopolysaccharide and glycolipid structural analysis.